16S V3-V4
Total nucleic acids were purified using the Amplilute Liquid Media Extraction Kit (Roche Molecular Diagnostics, Milan, Italy), based on the employment of QIAamp MinElute Columns, following the manufacturers instructions. The elution step was performed using 120l of the AVE elution buffer provided within the kit. Bacterial 16S rRNA gene targeted sequencing was performed using the Quick-16S NGS Library Prep Kit (Zymo Research, Irvine, CA). The bacterial 16S primers to amplify the V3-V4 region of the 16S rRNA gene have been custom-designed by Zymo Research. The final pooled library was cleaned with the Select-a-Size DNA Clean & Concentrator (Zymo Research, Irvine, CA), then quantified with TapeStation (Agilent Technologies, Santa Clara, CA) and Qubit (Thermo Fisher Scientific, Waltham, WA). The final library was sequenced on Illumina MiSeq with a v3 reagent kit (600 cycles). The sequencing was performed with 10% PhiX spike-in.